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...e in oxidative stress resistance. Prolinemetabolism activates theOxyR regulon.The effect of proline on cell survival was next tested by using a panel ofmutants from the E. coli Keio strain collection =-=(42)-=-. The cell survival rates of BW25113 wild-type and mutant strains after H2O2 stress treatment were tested in the absence and presence of proline (Fig. 6A). FIG 4 Catalase expression and activity are u...

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...18 plasmid was described previously (5). E. coli strains used in this study are listed in Table 1. The MG1655 putA strain was generated in this work by P1 transduction of the MG1655 wild-type strain =-=(29)-=-. E. coli cultures were grown in Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) or glucose minimalmedium (0.5 g glucose, 0.1 g thiamine, 1mMMgSO4, 0.5 gNaCl, 1 gN...

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... that proline metabolism increases intracellular H2O2 levels. OxyR reacts with H2O2 to form a disulfide bond between Cys199 and Cys208, which results in transcriptional activation of the OxyR regulon =-=(52, 53)-=-. Oxidation of OxyR and activation of the OxyR regulon have been reported to occur with 0.05 to 0.2 M H2O2 (32, 54). We observed a significant increase in endogenous H2O2 levels in katG cells with p...

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...with the PutApUC18 vector. Next, we questioned the mechanism by which proline enhances resistance to H2O2. Scavenging enzymes, like peroxidases and catalases, are a key defense mechanism against H2O2 =-=(40)-=-. To test whether proline increased the scavenging of H2O2,which is membrane permeable, extracellular H2O2 levels (Fig. 3C) were measured in cultures ofMG1655wild-type andputA cells grown to exponent...

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... Cys199 and Cys208, which results in transcriptional activation of the OxyR regulon (52, 53). Oxidation of OxyR and activation of the OxyR regulon have been reported to occur with 0.05 to 0.2 M H2O2 =-=(32, 54)-=-. We observed a significant increase in endogenous H2O2 levels in katG cells with proline, and inwild-type cells, H2O2 levels were found to be0.1Mwith FIG 7 Proline metabolism generates H2O2. (A) B...

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...ciated with phagocytic killing of microbes (60). Previously, pretreatment of E. coli cells with small amounts of H2O2 was shown to have a protective effect by a 10-fold induction of hydroperoxidase I =-=(61, 62)-=-. Here, proline metabolismmay also provide a preconditioning effect by generating H2O2 as a by-product and elevating hydroperoxidase I levels, thereby increasing the overall stress tolerance of the ce...

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...ciated with phagocytic killing of microbes (60). Previously, pretreatment of E. coli cells with small amounts of H2O2 was shown to have a protective effect by a 10-fold induction of hydroperoxidase I =-=(61, 62)-=-. Here, proline metabolismmay also provide a preconditioning effect by generating H2O2 as a by-product and elevating hydroperoxidase I levels, thereby increasing the overall stress tolerance of the ce...

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...gulon to H2O2 stress (51). Previous work has addressedmetabolic sources of endogenous ROS in E. coli and indicated that the respiratory chain contributes to the majority of endogenous H2O2 production =-=(55)-=-. It was found, however, thatH2O2 can also be significantly generated inE. coli by enzymes not associatedwith the respiratory chain (56). The oxidation of proline by PutA provides reducing equivalents...

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... antibody against a polypeptide containing PutA residues 1 to 47 (5). H2O2 production assays. H2O2 produced and accumulated within cells passes throughmembranes and equilibrateswith the culturemedium =-=(32)-=-. To determine the effect of L-proline treatment on H2O2 production in vivo, BW25113 (Keio strain collection) wild-type and katG (strain JW3914) cells were grown to an OD600 of 0.3 in glucose minimal...

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...n Fig. 2C. The above-described experimentswere performed in LBbroth, which is abundant in L-proline (9.5mM) and contains low glucose (0.1 mM), necessitating E. coli to utilize amino acids for growth =-=(37)-=-. It was previously reported that L-proline is significantly utiFIG 2 Depletion of PutA increases oxidative stress sensitivity. (A) Disk assays were performed with CSH4 (parental wild type), JT31 (put...

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...minishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a property which has been explored in eukaryotes such as fungi, plants, and animals =-=(19, 20, 27, 28)-=-. Themechanism by which proline protects against oxidative stress appears to involve protection of intracellular redox homeostasis and, in the fungal pathogen Colletotrichum trifolii, upregulation of ...

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... 9), the CSH4 strain contains mutations in relA and spoT, which regulate (p)ppGpp levels and are important for bacterial survival under nutrient starvation and oxidative and osmotic stress conditions =-=(38, 39)-=-. Thus, to further evaluate the effects of proline metabolism on oxidative stress sensitivity, aputA strain ofMG1655 was generated by P1 transduction (Fig. 3B). MG1655 wild-type and putA strains exh...

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...minishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a property which has been explored in eukaryotes such as fungi, plants, and animals =-=(19, 20, 27, 28)-=-. Themechanism by which proline protects against oxidative stress appears to involve protection of intracellular redox homeostasis and, in the fungal pathogen Colletotrichum trifolii, upregulation of ...

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... that proline metabolism increases intracellular H2O2 levels. OxyR reacts with H2O2 to form a disulfide bond between Cys199 and Cys208, which results in transcriptional activation of the OxyR regulon =-=(52, 53)-=-. Oxidation of OxyR and activation of the OxyR regulon have been reported to occur with 0.05 to 0.2 M H2O2 (32, 54). We observed a significant increase in endogenous H2O2 levels in katG cells with p...

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...e did not protectkatG cells, indicating that hydroperoxidase I is necessary for proline-enhanced protection againstH2O2 stress in E. coli. The katG gene is regulated by the transcription factor OxyR =-=(50)-=-, which is a critical regulator of the cellular response to H2O2 and thiol redox changes. The OxyR regulon regulates response genes, such as katG, grxA (glutaredoxin I), trxC (thioredoxin 2), and ahpC...

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...uots were removed at different time intervals, and H2O2 was measured immediately by an Amplex Red hydrogen peroxide-peroxidase assay kit (Life Technology) at room temperature, as previously described =-=(31)-=-. Fluorescence measurements were made with an Agilent (Varian) Cary Eclipse fluorescence spectrophotometer with excitation at 545 nm and fluorescence emission monitoring at 590 nm. Real-time PCR. MG16...

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...ment of intracellular H2O2 in wild-type cells grown without proline is consistent with the physiological concentration of H2O2 (0.1M) reported previously for E. coli in the exponential growth phase =-=(59)-=-. Twofold increases in H2O2 production, which we observed with proline, have also been shown to significantly induce katG expression with intracellular H2O2 at 0.1 to 0.2 M (55). Thus, increases in t...

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...creasing the overall stress tolerance of the cell. Various studies of proline metabolism in eukaryotes have shown that proline oxidation, which in eukaryotes occurs in themitochondrion, generates ROS =-=(19, 23, 63, 64)-=-, which can mediate cell death (63), cell survival against oxidative stress (28), and life span (64). The results from this work further illustrate the fundamental importance of how H2O2, as a metabol...

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...lutamate by the NAD-dependent P5CDH domain (2). In certain Gram-negative bacteria such as Escherichia coli, PutA also has an N-terminal ribbon-helix-helix (RHH) DNA-binding domain (residues 1 to 47) =-=(4)-=-. The RHH domain enables PutA to act as an autogenous transcriptional regulator of the putA and putP (highaffinity Na-proline transporter) genes (4). PutA represses put gene expression by binding to ...

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...antage to bacteria in harsh oxidative environments. Bacteria often encounter oxidative stress from the host immune system, such as the respiratory burst associated with phagocytic killing of microbes =-=(60)-=-. Previously, pretreatment of E. coli cells with small amounts of H2O2 was shown to have a protective effect by a 10-fold induction of hydroperoxidase I (61, 62). Here, proline metabolismmay also prov...

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... that proline catabolismmay account for the differences in oxidative stress resistance observed between strains CSH4 and JT31. Although the CSH4 and JT31 strains are commonly used for proline studies =-=(4, 5, 9)-=-, the CSH4 strain contains mutations in relA and spoT, which regulate (p)ppGpp levels and are important for bacterial survival under nutrient starvation and oxidative and osmotic stress conditions (38...

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...otes, PRODHandP5CDHareseparatelyencodedenzymes localized in the mitochondrion. In Gram-negative bacteria, PRODH and P5CDH are combined into a bifunctional enzyme known as proline utilization A (PutA) =-=(1, 2)-=-. The PRODH domain contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor and couples the two-electron oxidation of proline to the reduction of ubiquinone in the cytoplasmicmembrane...

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...4). In H. pylori, L-proline is a preferred respiratory substrate in the gut, with proline levels 10fold higher in the gastric juice of patients infected with H. pylori than in noninfected individuals =-=(10, 15)-=-. AnH. pylori putAmutant strain was shown to be less efficient in the colonization of mice than the wild-type strain (16). A putAmutant strain of the closely related mouse pathogen Helicobacter hepati...

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... H2O2 formed min 1 mg1 of membrane protein. To determine the rate of intracellular H2O2 formation, we used the relationship that 1 ml of bacteria at 1.0 OD unit comprises 0.47l of cytosolic volume =-=(35)-=-. For these assays, 20mg of membrane protein vesicles was isolated from 500-ml cultures grown to 0.8 OD units, which corresponds to a total cytosolic volume of 188 l. The rate of intracellular H2O2 f...

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...nous transcriptional regulator of the putA and putP (highaffinity Na-proline transporter) genes (4). PutA represses put gene expression by binding to five operator sites in the put regulatory region =-=(5)-=-. Transcription of the put genes is activated by proline, which causes a reduction of the PutA flavin cofactor and subsequent localization of PutA on the membrane (5–9). Proline has been shown to be a...

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...putA cells, no significant change in catalase activity was observed with proline. E. coli has two catalases, hydroperoxidase I and hydroperoxidase II, which are encoded by katG and katE, respectively =-=(41)-=-. The expression of katE is regulated by RpoS and is upregulated during stationary phase, whereas the expression of katG is regulated by OxyR and is induced byH2O2 (41). Changes in expression levels o...

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.... Besides being an important energy source, proline also provides protective benefits against abiotic and biotic stresses in a broad range of organisms (18–23). Proline is a well-known osmoprotectant =-=(24, 25)-=-, and in E. coli, proline has been described as being a thermoprotectant by diminishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a prop...

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... glutamate is a four-electron oxida-tion process that is coordinated in two successive steps by the enzymes proline dehydrogenase (PRODH) and 1-pyrroline-5-carboxylate dehydrogenase (P5CDH) (Fig. 1) =-=(1)-=-. In eukaryotes, PRODHandP5CDHareseparatelyencodedenzymes localized in the mitochondrion. In Gram-negative bacteria, PRODH and P5CDH are combined into a bifunctional enzyme known as proline utilizatio...

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.... Besides being an important energy source, proline also provides protective benefits against abiotic and biotic stresses in a broad range of organisms (18–23). Proline is a well-known osmoprotectant =-=(24, 25)-=-, and in E. coli, proline has been described as being a thermoprotectant by diminishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a prop...

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...d in control assays without L-proline. PutA activity in the membrane vesicles was confirmed by quantifying P5C production (nmol P5C min1 mg1 of membrane protein) using o-AB, as previously described =-=(36)-=-. Statistical analysis. The reported mean values and standard deviations are from three to five experiments. Data were analyzed by Student’s t tests, with statistical significance considered to be a P...

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...ture of flavoenzymes. In a previous study, the reactivity of different PutA proteins with molecular oxygen was evaluated, and PutA fromE. coliwas shown to have a turnover rate of0.3min1 with oxygen =-=(58)-=-. Thus, we propose that endogenous H2O2 from prolinemetabolism is not generated directly by PutA but rather by PutA/PRODHmainly passing electrons into the ubiquinone pool and PutA/P5CDHproducingNADH, ...

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...roline. DISCUSSION Proline is a multifaceted amino acid with important roles in carbon and nitrogen metabolism; protein synthesis; and protection against various environmental factors such as drought =-=(44)-=-, metal toxicity (45, 46), osmotic stress (24, 25), ultraviolent irradiation (47), unfolded protein stress (26, 48), and oxidative stress (19, 23, 27, 28, 49). In this study, we explored the role of p...

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...arbon and nitrogen metabolism; protein synthesis; and protection against various environmental factors such as drought (44), metal toxicity (45, 46), osmotic stress (24, 25), ultraviolent irradiation =-=(47)-=-, unfolded protein stress (26, 48), and oxidative stress (19, 23, 27, 28, 49). In this study, we explored the role of proline metabolism in oxidative stress protection by characterizing the oxidative ...

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... The PRODH domain contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor and couples the two-electron oxidation of proline to the reduction of ubiquinone in the cytoplasmicmembrane =-=(3)-=-.Theproductof thePRODHreaction, 1-pyrroline-5-carboxylate (P5C), is subsequently hydrolyzed to glutamate--semialdehyde (GSA), which is then oxidized to glutamate by the NAD-dependent P5CDH domain (...

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...0 nm. The kinetics of H2O2 formation from proline were determined by using inverted membrane vesicles from the katG strain (JW3914). Inverted membrane vesicles were prepared, as described previously =-=(33)-=-, from JW3914 (katG) cells grown in minimal A medium [33 mM KH2PO4, 51 mM K2HPO4, 8 mM (NH4)2SO4, 0.4 mM MgSO4, 0.5 mM tryptophan, 10 mM L-proline, 8% glycerol, and 0.05% glucose] to an OD600 of 0.7....

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... 9), the CSH4 strain contains mutations in relA and spoT, which regulate (p)ppGpp levels and are important for bacterial survival under nutrient starvation and oxidative and osmotic stress conditions =-=(38, 39)-=-. Thus, to further evaluate the effects of proline metabolism on oxidative stress sensitivity, aputA strain ofMG1655 was generated by P1 transduction (Fig. 3B). MG1655 wild-type and putA strains exh...

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...chain contributes to the majority of endogenous H2O2 production (55). It was found, however, thatH2O2 can also be significantly generated inE. coli by enzymes not associatedwith the respiratory chain =-=(56)-=-. The oxidation of proline by PutA provides reducing equivalents directly to the respiratory pathway via ubiquinone (3). The PRODH domain of PutA contains a FAD cofactor that couples the oxidation of ...

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...n the membrane (oxidative half-reaction). The ratelimiting step in the proline:ubiquinone oxidoreductase reaction catalyzed by PutA is the oxidative step (reduced FAD [FADH2] oxidation by ubiquinone) =-=(57)-=-. The production of H2O2 by proline oxidation would conceivably involve increased flux in the respiratory chain or aberrant electron transfer from FADH2 to molecular oxygen, generating superoxide anio...

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...4). In H. pylori, L-proline is a preferred respiratory substrate in the gut, with proline levels 10fold higher in the gastric juice of patients infected with H. pylori than in noninfected individuals =-=(10, 15)-=-. AnH. pylori putAmutant strain was shown to be less efficient in the colonization of mice than the wild-type strain (16). A putAmutant strain of the closely related mouse pathogen Helicobacter hepati...

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...minishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a property which has been explored in eukaryotes such as fungi, plants, and animals =-=(19, 20, 27, 28)-=-. Themechanism by which proline protects against oxidative stress appears to involve protection of intracellular redox homeostasis and, in the fungal pathogen Colletotrichum trifolii, upregulation of ...

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...minishing protein aggregation during heat stress (26). Proline has also been found to help combat oxidative stress, a property which has been explored in eukaryotes such as fungi, plants, and animals =-=(19, 20, 27, 28)-=-. Themechanism by which proline protects against oxidative stress appears to involve protection of intracellular redox homeostasis and, in the fungal pathogen Colletotrichum trifolii, upregulation of ...

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...oline is a multifaceted amino acid with important roles in carbon and nitrogen metabolism; protein synthesis; and protection against various environmental factors such as drought (44), metal toxicity =-=(45, 46)-=-, osmotic stress (24, 25), ultraviolent irradiation (47), unfolded protein stress (26, 48), and oxidative stress (19, 23, 27, 28, 49). In this study, we explored the role of proline metabolism in oxid...

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...oline is a multifaceted amino acid with important roles in carbon and nitrogen metabolism; protein synthesis; and protection against various environmental factors such as drought (44), metal toxicity =-=(45, 46)-=-, osmotic stress (24, 25), ultraviolent irradiation (47), unfolded protein stress (26, 48), and oxidative stress (19, 23, 27, 28, 49). In this study, we explored the role of proline metabolism in oxid...

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... protein synthesis; and protection against various environmental factors such as drought (44), metal toxicity (45, 46), osmotic stress (24, 25), ultraviolent irradiation (47), unfolded protein stress =-=(26, 48)-=-, and oxidative stress (19, 23, 27, 28, 49). In this study, we explored the role of proline metabolism in oxidative stress protection by characterizing the oxidative stress response of wild-type and p...

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... juice of patients infected with H. pylori than in noninfected individuals (10, 15). AnH. pylori putAmutant strain was shown to be less efficient in the colonization of mice than the wild-type strain =-=(16)-=-. A putAmutant strain of the closely related mouse pathogen Helicobacter hepaticus also exhibited less pathogenicity in mice than the wild-type strain (17). Thus, in certain ecological niches, PutA an...

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...ion against various environmental factors such as drought (44), metal toxicity (45, 46), osmotic stress (24, 25), ultraviolent irradiation (47), unfolded protein stress (26, 48), and oxidative stress =-=(19, 23, 27, 28, 49)-=-. In this study, we explored the role of proline metabolism in oxidative stress protection by characterizing the oxidative stress response of wild-type and putAmutant E. coli strains.Wildtype E. coli ...

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...nge of organisms (18–23). Proline is a well-known osmoprotectant (24, 25), and in E. coli, proline has been described as being a thermoprotectant by diminishing protein aggregation during heat stress =-=(26)-=-. Proline has also been found to help combat oxidative stress, a property which has been explored in eukaryotes such as fungi, plants, and animals (19, 20, 27, 28). Themechanism by which proline prote...

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...imal medium to an OD600 of 0.3 before treatment with 10 mM L-proline and L-THFA. Samples were collected at designated time points, and -galactosidase activities were measured as described previously =-=(30)-=-. To determine the effect of H2O2 on katG expression, cells were cultured as described above and then treated with different concentrations of H2O2 for 30 min, followed bymeasurement of -galactosidas...

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...n contrast, proline increased the survival rates ofkatE andsoxR cells, indicating that hydroperoxidase II and SoxR, which is a transcription factor activated in response to redox-active metabolites =-=(43)-=-, are not essential for the mechanism of proline protection. Besides H2O2 scavenging, activation of OxyR initiates other oxidative stress systems, such as the sequestration of unincorporated iron by D...

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...creasing the overall stress tolerance of the cell. Various studies of proline metabolism in eukaryotes have shown that proline oxidation, which in eukaryotes occurs in themitochondrion, generates ROS =-=(19, 23, 63, 64)-=-, which can mediate cell death (63), cell survival against oxidative stress (28), and life span (64). The results from this work further illustrate the fundamental importance of how H2O2, as a metabol...